Phospholipid composition and microbubbles and emulsions formed using same

ABSTRACT

A composition, and method thereof, for stabilizing a fluorocarbon emulsion includes phosphatidylcholine, phosphatidylethanolamine-PEG, and a cone-shaped lipid.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims priority to a United States Provisionalapplication filed Jun. 12, 2014 and having Ser. No. 62/011,469, whereinthat Provisional application is hereby incorporated herein by reference.

FIELD OF THE INVENTION

A phospholipid composition is disclosed. In certain embodiments, thephospholipid composition can be used as an ultrasound contrast agent(“USCA”), where that USCA comprises improved shelf-life and patienttolerability.

BACKGROUND OF THE INVENTION

Ultrasound contrast agents (USCA) are used for improving diagnosticaccuracy on ultrasound imaging. USCA have also been used as cavitationnuclei for therapeutic procedures such as sonothrombolysis useful fortreating stroke and heart attack. At the current time, however, the mainuse of USCA is for diagnosis.

A prior art ultrasound contrast agent is sold in commerce under thetrademark DEFINITY. DEFINITY is a phospholipid-based ultrasound contrastagent comprising dipalmitoylphosphatidylcholine (“DPPC”),dipalmitoylphosphatidylethanolamine-PEG(5,000) (“DPPE-PEG5,000”), anddipalmitoylphosphatidic acid (“DPPA”).

DEFINITY has a shelf-life of two-years at 4-8° C. Hydrolysis of thelipids is primarily responsible for degradation of the product. Clinicaluse of DEFINITY is known to cause back pain as a side-effect. Theprescribing information for DEFINITY expressly discloses that back painoccurs in about 1.2% of patients. When such back pain does occur, thatside effect can be very unpleasant for the patient and last up to 30minutes or one hour.

SUMMARY OF THE INVENTION

Applicants' composition comprises a plurality of lipids that issubstantially charge neutral at neutral pH, i.e. pH=7.0 useful forstabilizing emulsion and microbubbles of fluorocarbons. The formulationcomprises phosphatidylcholine with a PEG′ylated lipid and a third lipidwhich is a cone-shaped lipid. In certain embodiments, the cone-shapedlipid is phosphatidylethanolamine. The formulation can generateemulsions and microbubbles that show enhanced stability to storage andshow propensity to lessened side-effects. In certain embodiments,Applicants' composition may also include a fourth lipid which is abifunctional PEG′ylated lipid. Because microbubbles and emulsionnanoparticles prepared with Applicants' composition are overallcharge-neutral, those microbubbles/that emulsion comprises enhancedproperties for targeting biologically relevant epitopes and biomarkers.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from a reading of the followingdetailed description taken in conjunction with the drawings in whichlike reference designators are used to designate like elements, and inwhich:

FIG. 1 shows the ionization of dipalmitoylphosphatidic acid at varyingpH;

FIG. 2 illustrates a cone-shaped lipid and a cylindrical-shaped lipid;

FIG. 3 graphically illustrates particle sizing for two (2) differentformulations;

FIG. 4 graphically illustrates total microbubble counts for compositionscontaining cholesterol;

FIG. 5 graphically illustrates total microbubble counts for compositionscontaining palmitic acid;

FIG. 6 graphically illustrates particle count versus particle diameterfor MVT-100 and a DEFINITY EQUIVALENT;

FIG. 7 graphically illustrates total microbubble counts versus DEFINITYEQUIVALENT for different ratios of diluents;

FIG. 8 graphically illustrates total microbubble counts versus DEFINITYEQUIVALENT for different ratios of diluents;

FIG. 9 graphically illustrates total microbubble counts versus DEFINITYEQUIVALENT for different ratios of diluents;

FIG. 10 graphically illustrates percent total microbubble counts versusDEFINITY EQUIVALENT for three different lipid ratios with differentdiluents;

FIG. 11 graphically illustrates stability data for a DEFINITY EQUIVALENTcomprising DPPA and for MVT-100 comprising DPPE instead of DPPA;

FIG. 12 graphically illustrates total microbubble counts measured fortwo different formulations wherein each formulation includes a cationiclipid in combination with a cone-shaped lipid and a third formulationwhere the cationic lipid replaces DPPE as the cone-shaped lipid;

FIG. 13 graphically illustrates microbubble counts for each of theformulations recited versus Draw# at four (4) minutes post activation;

FIG. 14 graphically illustrates microbubble sizing data for the 82 molepercent DPPC, 10 mole percent DSTAP, and 8 mole percent DPPE-MPEG-5Kformulation at four (4) minutes post activation;

FIG. 15 graphically illustrates microbubble counts for each of theformulations recited versus Draw# at sixty-four (64) minutes postactivation;

FIG. 16 graphically illustrates microbubble sizing data for the 82 molepercent DPPC, 10 mole percent DSTAP, and 8 mole percent DPPE-MPEG-5Kformulation at sixty-four (64) minutes post activation; and

FIG. 17 graphically shows ultrasound imaging data relating to residualmicrobubbles in the renal cortices, where a Definity equivalentaccumulated over three-fold more residual microbubbles in the renalcortices than did MVT-100.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention is described in preferred embodiments in the followingdescription with reference to the Figures, in which like numbersrepresent the same or similar elements. Reference throughout thisspecification to “one embodiment,” “an embodiment,” or similar languagemeans that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the invention. Oneskilled in the relevant art will recognize, however, that the inventionmay be practiced without one or more of the specific details, or withother methods, components, materials, and so forth. In other instances,well-known structures, materials, or operations are not shown ordescribed in detail to avoid obscuring aspects of the invention.

In certain embodiments, Applicant's phospholipid composition comprisesone or more substantially charge-neutral phospholipids, wherein lipidcoated microbubble-forming emulsions comprising Applicant's phospholipidcomposition comprise improved stability on storage, and lipid coatedmicrobubbles formed from Applicant's microbubble-forming emulsions whenused clinically are associated with a decrease in bioeffects, e.g. backpain. In certain embodiments, one or more of Applicants' phospholipidscomprises a zwitterionic compound which is overall charge-neutral.

In certain embodiments, Applicant's phospholipid composition comprisesdipalmitoylphosphatidylcholine (“DPPC”), phospholipid 1. DPPC is azwitterionic compound, and is a substantially neutral phospholipid.

In certain embodiments, Applicants' phospholipid composition comprises asecond phospholipid 2 comprising a polyhydroxy head group, and/or aheadgroup of greater than 350 Daltons, wherein M⁺ is selected from thegroup consisting of Na⁺, K⁺, Li⁺, and NH₄ ⁺.

In certain embodiments, Applicant's phospholipid 2 comprisesphospholipid 3 comprising a sodium cation and a glycerol headgroup boundto the phosphoryl moiety.

Phospholipid 4 comprises an ammonium counterion and a polyethyleneglycol (“PEG”) headgroup bound to the phosphoryl moiety. In certainembodiments, Applicants' composition comprises a PEG′ylated lipid. Incertain embodiments, the PEG group MW is from about 1,000 to about10,000 Daltons. In certain embodiments, the PEG group MW is from about2,000 to about 5,000 Daltons. In certain embodiments, the PEG group MWis about 5,000 Daltons.

In certain embodiments, Applicants' lipid composition includes one ormore of the following PEG′ylated lipids:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000] (ammonium salt),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000] (ammonium salt),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000] (ammonium salt),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (ammonium salt),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phospboethanolamine-N-[methoxy(polyethyleneglycol)-3000] (ammonium salt),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000](ammonium salt),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphothanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt)

Phospholipid 5, shown above, representsdipalmitoylphosphatidylethanolamine, or DPPE. PE, particularly DPPE is apreferred lipid in the invention, preferably in the formulation with theother lipids at concentration of between 5 and 20 mole percent, mostpreferably 10 mole percent.

In certain embodiments, Applicant's phospholipid composition includes noPhosphatidic acid 6 (“DPPA”).

As those skilled in the art will appreciate, DPPA comprises two acidicprotons. The pKa for the second acidic proton is about 7.9. FIG. 1graphically depicts the ionization of DPPA as a function of pH. Curve110 recites the percent of mono-anion 6 present, and curve 120 recitesthe percent of di-anion 7 present. At a pH of about 4 and higher, thecombined percentages of mono-anion 6 and di-anion 7 total 100.

At a pH of about 7.9, DPPA comprises about 50 percent mono-anion 7 andabout 50 percent di-anion 8. At a pH of about 7.0, DPPA comprises about85 percent mono-anion 7 and about 15 percent di-anion 8.

Phosphatidic acid DPPA plays several roles in the functioning of cells;being utilized as a precursor in the biosynthesis of other lipids,facilitating vesicle fission/fusion via its biophysical properties andacting as a signaling lipid. The monoacyl derivative, lysophosphatidicacid (LPA), acts as a potent signaling molecule through the activationof high-affinity G-protein coupled receptors (LPA₁, LPA₂ and LPA₃,formerly, EDG₂, EDG₄ and EDG₇; and recently identified LPA₄, LPA₅ andLPA₆). As prior art phospholipid compositions comprising DPPA, such asfor example DEFINITY, age and DPPA undergoes hydrolysis, the presence ofthe monoacyl derivative likely increases, with a clinical increase inundesirable bioeffects.

Table 1 shows the stability of a phospholipid composition comprisingDPPA on storage at 4-8° C. The ratios of the individual lipid componentsare the ratios utilized in the prior art DEFINITY product. At 38 monthsof cold temperature storage DPPC is still 86.4% of its release level.DPPE-PEG 5,000 is 81.6% of its release level and DPPA is 78.4% of itsrelease level.

At 48 months, however, the DPPA falls below its specification while DPPCand DPPE-PEG remained within specifications. Applicant has found thatthe phosphatidic acid DPPA is the limiting factor with respect to thecold storage stability of a phospholipid composition comprising aplurality of phospholipids in combination with DPPA. Furthermore,applicant has discovered that the other lipids are more stable informulations without DPPA; it appears that DPPA catalyzes or acceleratesthe hydrolysis of the lipids in the formulation.

TABLE 1 Time Points Shelf-Life 14 38 Test Parameter SpecificationsRelease Months Months Appearance (n = 6) Uniformly clear to Pass PassPass translucent, colorless liquid Particulates (n = 6) Free of visibleparticles Pass Pass Pass pH (n = 2) 5.5-7.5 7.00 7.01 7.02Octafluoropropane Assay (mg/ml) (n = 5) ≥5.5 mg/ml 7.60 6.91 6.619 LipidAssay DPPC 0.864-1.296 1.116 1.090 0.964 (mg/ml) (n = 2) DPPE-MPEU 50000.640-0.960 0.881 0.815 0.719 DPPA 0.096-0.144 0.125 0.109 0.098 TotalLipid (mg ml) 1.60-2.40 2.12 2.01 1.781 Size Distribution 0.56 μm to1.06 μm report only 2.65E+10 1.95E+10 8.21E+09 (particles/ml) 1.06 μm to2.03 μm ≥1.0 × 10⁸ 8.10E+09 6.96E+09 3.96E+09 (n = 6) 2.03 μm to 5.99 μm≥1.0 × 10⁷ 6.00E+08 4.38E+08 2.62E+08 5.99 μm to 10.27 μm report only6.96E+07 4.96E+07 1.75E+07 ≥10.27 μm ≤5.0 × 10⁸ 4.40E+06 4.20E+062.85E+06 total report only 3.52E+10 2.69E+10 1.24E+10 Endotoxin ≤80EU/vial Pass NR NR Sterility Sterile Pass NR Pass

DPPA was incorporated into prior art phospholipids-based imaging agentsto prevent possible aggregation of microbubbles. The di-anionicstructure of DPPA resulted in increase electrostatic repulsion oflipid-coated microbubbles, thereby, it was thought reducing thelikelihood of microbubble aggregation.

Surprisingly, Applicant has discovered that lipid-coated microbubblesprepared from a plurality of phospholipids but without DPPA do notundergo the undesirable aggregation. Moreover, lipid-coated microbubblesprepared from one or more phospholipids but without DPPA have similarparticle size as DEFINITY.

In certain embodiments, Applicant's phospholipid composition comprisesan injectable suspension. A vial for Applicant's injectable composition,upon activation, yields a plurality of phospholipid coated microspheresencapsulating a fluorocarbon gas. Such phospholipid-coated microspherescomprise a diagnostic drug that is intended to be used for contrastenhancement during certain indicated echocardiographic procedures.

Applicant's phospholipid composition comprises a clear, colorless,sterile, non-pyrogenic, hypertonic liquid, which upon activationprovides a homogeneous, opaque, milky white injectable suspension ofphospholipid coated microspheres encapsulating a fluorocarbon gas. Incertain embodiments, that suspension is administered by intravenousinjection.

Referring now to FIG. 2, in certain embodiments Applicants' inventioncontains one or more cone shaped or hexagonal HII forming lipids.Cone-shaped lipids, such as lipid 210, useful in the invention includemonogalactosyldiacylglycerol (MGDG), monoglucosyldiacylglycerol (MGDG),diphosphatidylglycerol (DPG) also called cardiolipin, phosphatidylserine(PS), phosphatidylethanolamine (PE) and diacylglycerol. Phosphatidicacid (PA) is also a cone-shaped lipid, but is not preferred due to itspropensity to hydrolysis and potential to cause bioeffects. The mostpreferred cone-shaped phospholipid is phoshatidylethanolamine (PE).

Cone shaped lipid 210 comprises a head group 212 that occupies a smallervolume than do the pendent groups 214 extending outwardly from headgroup 212. Cylindrical-shaped lipid 220 comprises a head group 222 thatoccupies a similar volume as that volume defined by the pendent groups224 extending outwardly from head group 222. In addition the applicantshave discovered that cationic, i.e. positively charged lipids can beused as cone shaped lipids provided that the head group of said cationiclipid is smaller than the tail.

Examples of potentially useful cone-shaped cationic lipids include butare not limited to 1,2-dioleoyl-3-trimethylammonium-propane (chloridesalt), 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate salt),1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt),1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt),1,2-distearoyl-3-trimethylammonium-propane (chloride salt),1,2-dioleoyl-3-dimethylammonium-propane,1,2-dimyristoyl-3-dimethylammonium-propane,1,2-dipalmitoyl-3-dimethylammonium-propane,1,2-distearoyl-3-dimethylammonium-propane, Dimethyldioctadecylammoniumand 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt),O,O-di-O-octadecenyl-3-tα-trimethylammonioacetyl-diethanolamine.

Referring now to FIG. 3, the inventors have discovered that microbubblesprepared with a third lipid—a cone-shaped lipid, in particular DPPE,provide better bubble count and better microbubble stability thanformulations without such a third lipid. Preferably the cone-shapedlipid is provided within the formulation at a concentration of betweenabout 5 and about 20 mole percent and more preferably at about 8 to 15mole percent and most preferably at about 10% of the total lipid in theformulation.

As shown in FIG. 3, formulations without DPPA, and without a cone-shapedlipid. For example, the formulation comprising a lipid composition at0.75 mg/ml generates few microbubbles. The formulation comprising alipid composition at 1.50 mg/ml failed to generate microbubble particlecounts similar to the Definity Equivalent.

In certain embodiments, Applicant's phospholipid composition comprisesoctafluoropropane encapsulated in an outer lipid shell consisting of(R)-4-hydroxy-N,N,N-trimethyl0-oxo-7-[(1-oxohexadecyl)oxy]-3,4,9-trioxa-4-phosphapentacosan-1-aminium,4-oxide, inner salt, i.e. DPPC, and(R)-α-[6-hydroxy-6-oxido-9-[(l-oxohexadecyl)oxy]5,7,11-trioxa-2-aza-6-phosphahexacos-1-yl]-ω-methoxypoly(ox-1,2-ethanediyl),monosodium salt, i.e. DPPE PEG5000/Phospholipid 4 with lipid 5, DPPE.DPPE-PEG5000 has an approximate molecular weight of 5750 Daltons.

Each mL of the clear liquid contains 0.75 mg lipid blend (consisting ofconsisting of 0.046 mg DPPE, 0.400 mg DPPC, and 0.304 mg MPEG5000 DPPE),103.5 mg propylene glycol, 126.2 mg glycerin, 2.34 mg sodium phosphatemonobasic monohydrate, 2.16 mg sodium phosphate dibasic heptahydrate,and 4.87 mg sodium chloride in Water for Injection. The pH is between6.2-6.8.

After activation, each mL of Applicant's phospholipid coatedmicrospheres encapsulating a fluorocarbon gas comprise a milky whitesuspension consisting essentially of a maximum of 1.2×10¹⁰ lipid-coatedmicrospheres, and about 150 microL/mL (1.1 mg/mL) octafluoropropane. Themicrosphere particle size parameters are listed below, and are identicalto those of DEFINITY:

Mean Particle Size 1.1-3.3 μm Particles Less than 10 μm 98% MaximumDiameter 20 μm

A comparison of the quantitative composition of Applicant's phospholipidcomposition and the prior art DEFINITY products is shown in TABLE 2,below.

In certain embodiments, Applicant's phospholipid composition isidentical to DEFINITY, with the exception that Applicant's phospholipidcomposition does not include any DPPA, but that an equimolar amount ofDPPE has been substituted for DPPA. Other components of the lipid blend(DPPC, DPPE PEG5000 and DPPe) have been proportionately increased tomaintain the total lipid blend at 0.75 mg.

TABLE 2 APPLICANT'S COMPOSITION DEFINITY OctafluoropropaneOctafluoropropane Pre activation: in vial headspace Pre activation: invial headspace Post activation: 1.1 mg/mL in Post activation: 1.1 mg/mLin lipid microspheres lipid microspheres 0.75 mg lipid blend (consisting0.75 mg lipid blend (consisting of 0.046 mg DPPE, 0.400 mg of 0.045 mgDPPA, 0.401 mg DPPC, and 0.304 mg MPEG5000 DPPC, and 0.304 mg MPEG5000DPPE) DPPE) 103.5 mg propylene glycol 103.5 mg propylene glycol 126.2 mgglycerin 126.2 mg glycerin 2.34 mg sodium phosphate 2.34 mg sodiumphosphate monobasic monohydrate monobasic monohydrate 2.16 mg sodiumphosphate 2.16 mg sodium phosphate dibasic heptahydrate dibasicheptahydrate 4.87 mg sodium chloride 4.87 mg sodium chloride Adjust pHto 6.2-6.8 with Adjust pH to 6.2-6.8 with NaOH or HCl NaOH or HCl Qs to1 mL Qs to 1 mL

For the fourth lipid, a bifunctional PEG′ylated lipid may be employed.Bifunctional PEG′ylated lipids include but are not limited toDSPE-PEG(2000) Succinyl1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethyleneglycol)-2000] (ammonium salt), DSPE-PEG(2000) PDP1,2-distearoly-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethyleneglycol)-2000] (ammonium salt), DSPE-PEG(2000) Maleimide1,2-distearoly-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt), DSPE-PEG(2000) Biotin1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt), DSPE-PEG(2000) Cyanur1,2-distearoly-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethyleneglycol)-2000] (ammonium salt), DSPE-PEG(2000) Amine1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt), DPPE-PEG(5,000)-maleimide,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethyleneglycol)-2000](ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-2000] (ammonium salt),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (ammonium salt),N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)5000]}.

The bifunctional lipids may be used for attaching antibodies, peptides,vitamins, glycopeptides and other targeting ligands to the microbubbles.The PEG chains MW may vary from about 1,000 to about 5,000 Daltons inthe third lipid. In certain embodiments, the PEG chains MW are fromabout 2,000 to about 5,000 Daltons.

The lipid chains of the lipids used in the invention may vary from about14 to about 20 carbons in length. Most preferably the chain lengths arefrom about 16 to about 18 carbons. Chains may be saturated orunsaturated but are preferably saturated. Cholesterol and cholesterolderivatives may also be employed in the invention with the proviso thatthey be neutral, or if negatively charged contain a head group greaterthan about 350 MW in juxtaposition to the negative charge to shield thecharge from the biological milieu.

In various embodiments, the microbubble core gas is nitrogen, oxygen,sulfur hexafluoride, perfluoropropane, perfluorobutane,perfluoropentane, perfluorohexane or mixtures thereof. For the purposesof imaging and drug delivery, the ideal microbubble core gas has lowaqueous solubility coupled with a boiling point below body temperature.This results in a microbubble with a long circulation time, a longuseful life span, and high echogenic qualities.

Applicant's gaseous precursors include, for example, fluorinatedcarbons, perfluorocarbons, sulfur hexafluoride, perfluoro ethers andcombinations thereof. As the stilled artisan will appreciate, aparticular fluorinated compound, such as sulfur hexafluoride, aperfluorocarbon or a perfluoro ether, may exist in the liquid state whenthe compositions are first made, and are thus used as a gaseousprecursor. Whether the fluorinated compound is a liquid generallydepends on its liquid/gas phase transition temperature, or boilingpoint. For example, a preferred perfluorocarbon, perfluoropentane, has aliquid/gas phase transition temperature (boiling point) of 29.5° C. Thismeans that perfluoropentane is generally a liquid at room temperature(about 25° C.), but is converted to a gas within the human body, thenormal temperature of which is about 37° C., which is above thetransition temperature of perfluoropentane. Thus, under normalcircumstances, perfluoropentane is a gaseous precursor. As known to oneskilled in the art, the effective boiling point of a substance may berelated to the pressure to which that substance is exposed. Thisrelationship is exemplified by the ideal gas law; PV=nRT, where P ispressure, V is volume, n is moles of substance, R is the gas constant,and T is temperature in ° K. The ideal gas law indicates that aspressure increases, the effective boiling point also increases.Conversely, as pressure decreases, the effective boiling pointdecreases.

Fluorocarbons for use as gaseous precursors in the compositions of thepresent invention include partially or fully fluorinated carbons,preferably perfluorocarbons that are saturated, unsaturated or cyclic.The preferred perfluorocarbons include, for example, perfluoromethane,perfluoroethane, perfluoropropane, perfluorocyclopropane,perfluorobutane, perfluorocyclobutane, perfluoropentane,perfluorocylcopentane, perfluorohexane, perfluorocyclohexane, andmixtures thereof. More preferably, the perfluorocarbon isperfluorohexane, perfluoropentane, perfluoropropane or perfluorobutane.

Preferred ethers include partially or fully fluorinated ethers,preferably perfluorinated ethers having a boiling point of from about36° C. to about 60° C. Fluorinated ethers are ethers in which one ormore hydrogen atoms is replaced by a fluorine atom. Preferredperfluorinated ethers for use as gaseous precursors in the presentinvention include, for example, perfluorotetrahydropyran,perfluoromethyltetrahydrofuran, perfluorobutylmethyl ether (e.g.,perfluoro t-butylmethyl ether, perfluoro isobutyl methyl ether,perfluoro n-butyl methyl ether), perfluoropropylethyl ether (e.g.,perfluoro isopropyl ethyl ether, perfluoro n-propyl ethyl other),perfluorocyclobutylmethyl ether, perfluorocyclopropylethyl ether,perfluoropropylmethyl ether (e.g., perfluoro isopropyl methyl ether,perfluoro n-propyl methyl ether), perfluorodiethyl ether,perfluorocyclopropylmethyl ether, perfluoromethylethyl ether andperfluorodimethyl ether.

Other preferred perfluoroether analogues contain between 4 and 6 carbonatoms, and optionally contain one halide ion, preferably Br—. Forexample, compounds having the structure Cn Fy Hx OBr, where n is aninteger of from 1 to about 6, y is an integer of from 0 to about 13, andx is an integer of from 0 to about 13, are useful as gaseous precursors.

Other preferable fluorinated compounds for use as gaseous precursors inthe present invention are sulfur hexafluoride and heptafluoropropane,including 1,1,1,2,3,3,3-heptafluoropropane and its isomer,1,1,2,2,3,3,3-heptafluoropropane. Mixtures of different types ofcompounds, such as mixtures of a fluorinated compound (e.g., aperfluorocarbon or a perfluoroether) and another type of gas or gaseousprecursor can also be used in the compositions of the present invention.Other gases and gaseous precursors are well known to one skilled in theart.

Generally, preferred gaseous precursors undergo phase transition to gasat a temperature up to about 57° C., preferably from about 20° C. toabout 52° C., preferably from about 37° C., to about 50° C., morepreferably from about 38° C. to about 48° C., even more preferably fromabout 38° C. to about 46° C., still even more preferably from about 38°C. to about 44° C., even still more preferably from about 38° C., toabout 42° C. Most preferably, the gaseous precursors undergo a phasetransition at a temperature of about less than 40° C. As will berecognized by one skilled in the art, the optimal phase transitiontemperature of a gaseous precursor for use in a particular applicationwill depend upon considerations such as, for example, the particularpatient, the tissue being targeted, the nature of the physiologicalstress state (i.e., disease, infection or inflammation, etc.) causingthe increased temperature, the stabilizing material used, and/or thebioactive agent to be delivered.

Additionally, one skilled in the art will recognize that the phasetransition temperature of a compound may be affected by local conditionswithin the tissue, such as, for example, local pressure (for example,interstitial, interfacial, or other pressures in the region). By way ofexample, if the pressure within the tissues is higher than ambientpressure, this will be expected to raise the phase transitiontemperature. The extent of such effects may be estimated using standardgas law predictions, such as Charles' Law and Boyle's Law. As anapproximation, compounds having a liquid-to-gas phase transitiontemperature between about 30° C. and about 50° C. can be expected toexhibit about a 1° C. increase in the phase transition temperature forevery 25 mm Hg increase in pressure. For example, the liquid-to-gasphase transition temperature (boiling point) of perfluoropentane is29.5° C. at a standard pressure of about 760 mm Hg, but the boilingpoint is about 30.5° C. at an interstitial pressure of 795 mm Hg.

Materials used in stabilizing the gaseous precursor, discussed herein,may also affect the phase transition temperature of the gaseousprecursor. In general, the stabilizing material is expected to increasethe phase transition temperature of the gaseous precursor. Inparticular, a relatively rigid polymeric material, such as, for example,polycyanomethacrylate, may have a significant effect on the phasetransition temperature of the gaseous precursor. Such an effect must beconsidered in the selection of the gaseous precursor and the stabilizingmaterial.

The gaseous precursors and/or gases are preferably incorporated in thestabilizing materials and/or vesicles irrespective of the physicalnature of the composition. Thus, it is contemplated that the gaseousprecursors and/or gases may be incorporated, for example, in stabilizingmaterials in which the stabilizing materials are aggregated randomly,such as emulsions, dispersions or suspensions, as well as in vesicles,including vesicles which are formulated from lipids, such as micellesand liposomes. Incorporation of the gases and/or gaseous precursors inthe stabilizing materials and/or vesicles may be achieved by using anyof a number of methods.

The terms “stable” or “stabilized” mean that the vesicles may besubstantially resistant to degradation, including, for example, loss ofvesicle structure or encapsulated gas, gaseous precursor and/orbioactive agent, for a useful period of time. Typically, the vesiclesemployed in the present invention have a desirable shelf life, oftenretaining at least about 90% by volume of its original structure for aperiod of at least about two to three weeks under normal ambientconditions. In preferred form, the vesicles are desirably stable for aperiod of time of at least about 1 month, more preferably at least about2 months, even more preferably at least about 6 months, still morepreferably about eighteen months, and yet more preferably up to about 3years. The vesicles described herein, including gas and/or gaseousprecursor filled vesicles, may also be stable even under adverseconditions, such as temperatures and pressures which are above or belowthose experienced under normal ambient conditions.

Useful gases in the invention are shown in Table 3 below.

TABLE 3 Molecular Aqueous Solubility Boiling Compound Weight (Ostwald'sCoefficient) Point ° C. Nitrogen 28 18071 −196 Oxygen 32 4865 −183Sulfur Hexafluoride 146 5950 −64 Perfluoropropane 188 583 −36.7Perfluorobutane 238 <500 −1.7 Perfluoropentane 288 >24 and <500 29Perfluorohexane 338 24 56.6

The following examples are presented to further illustrate to personsskilled in the art how to make and use the invention. These examples arenot intended as a limitation, however, upon the scope of the invention.

Example 1

A blend of lipids was prepared by suspending a mixture of lipidscontaining DPPC and DPPE-MPEG-5000 in propylene glycol. The lipidsuspension was heated to 65±50 C until dissolution of the lipids in thepropylene glycol was complete. The lipid solution was then added to anaqueous solution containing sodium chloride, phosphate buffer andglycerol and allowed to mix completely with gentle stirring. Each ml ofthe resultant lipid blend contained 0.75 mg total lipid (consisting of0.43 mg DPPC, and 0.32 mg DPPE-MPEG-5000). Each ml of the lipid blendalso contained 103.5 mg propylene glycol, 126.2 mg glycerin, 2.34 mgsodium phosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasicheptahydrate, and 4.87 mg sodium chloride in Water for Injection. The pHwas 6.2-6.8. The material was provided in scaled vials with a headspacecontaining octafluoropropane (OFP) gas (>80%) with the balance air.

Determination of the concentration and size distribution of themicrobubbles produced by formulations listed in both previous andsubsequent parts of this application were done in the following manner.Vials were activated using a Vialmix modified dental amalgamator andallowed to sit for 4 minutes before diluting a small amount of themicrobubble suspension with filtered normal saline in a suitablecontainer. After activation and dilution (1e-6) of the microbubblesolution, microbubble size distributions were determined using a Nicomp780 (Particle Sizing Systems) sampling in 128 channels. Microbubbleparticle sizing results obtained from lipid formulations listed in thisapplication were compared with a Definity Equivalent standard. Thisstandard contains approximately 82 mol % DPPC, 10 mol % DPPA, and 8 mol% DPPE-MPEG-5000 dissolved in the same buffered co-solvent salinemixture as the neutral formulation listed in Paragraph [00059].

Example 2—Preparation of Different Formulations

As shown in Table 4, the mole percent ratios of DPPC:DPPE-MPEG-5K wereadjusted from 91.16:8.84 to 94.00:6.00 at 0.75 mg/ml total with92.55:7.45 being the most stable, as shown in Table 4. Stability wasbased on the opacity of the vials after activation.

TABLE 4 FORM DPPC MPEG-5K-DPPE TOTAL LIPID 01 91.16 8.84 0.75 (mg/ml) 0292.00 8.00 0.75 (mg/ml) 03 92.55 7.44 0.75 (mg/ml) 04 94.00 6.00 0.75(mg/ml)

Additionally the volume percentages of propylene glycol and glycerolwere also adjusted from 0 to 20% in the most stable of two lipid blends;this had no effect on microbubbles stability.

Lipid blends containing cholesterol are shown in Table 5 and Table 6.Lipid blend formulations containing cholesterol, DPPC and DPPE-MPEG-5000at 0.75 and 1.50 mg/ml total lipid, produced lower concentrations ofmicrobubbles than did the Definity standard formulation (FIG. 4). Of thefour formulations containing cholesterol, the lipid blend with 81 mol %DPPC, 11 mol % DPPE-MPEG-5000, and 8% cholesterol produced the highestconcentration of microbubbles.

Referring to FIG. 5, formulations containing palmitic acid, DPPC, andDPPE-MPEG-5000 consistently produced higher concentrations ofmicrobubbles than did formulations containing only DPPC andDPPE-MPEG-5000. When compared against the Definity standard, theseformulations produced a higher concentration of larger-sized bubblesthat could pose a health risk. Other ingredients were added to the twolipid blend to optimize the concentration and size distribution of themicrobubbles. These excipients included stearic acid, Pluronic F68, and1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG). Formulationscontaining DSPG and stearic acid produced a higher concentration ofmicrobubbles than formulations containing DPPC and DPPE-MPEG-5000,however not to the extent that formulations containing DPPE were able to(See Example 3). The addition of Pluronic F68 to the two lipid blend didnot significantly increase the concentration of microbubbles.

TABLE 5 MOL % LIPID Microbubble DPPE- Choles- Mean Size Form DPPCMPEG-5K terol Vol Wt Num Wt 13 80.93 7.88 10.55 0.75 mg/ml 76.28 1.09 1881.53 7.92 10.57 1.50 mg/ml 77.93 1.58 20 77.22 7.45 15.32 1.50 mg/ml38.54 1.80 22 77.22 7.45 15.32 0.75 mg/ml 435.67 1.87

TABLE 6 MOL % LIPID Microbubble DPPE- Choles- Mean Size Form DPPCMPEG-5K terol Vol Wt Num Wt 17 80.93 7.88 11.19 0.75 mg/ml 432.46 0.8919 80.93 7.88 11.19 1.50 mg/ml 23.04 0.89 21 73.16 6.61 20.23 0.75 mg/ml12.60 0.88 23 73.16 6.61 23.23 1.50 mg/ml 389.41 0.93

Example 3 Preparation of MVT-100 (Formulation Containing DPPE)

A blend of lipids containing DPPC, DPPE and DPPE-MPEG-5000 was preparedusing similar methods to those listed in Example 1. The lipids,suspended in propylene glycol, were heated to 70±50 C until theydissolved. The lipid solution was then added to an aqueous solutioncontaining sodium chloride, phosphate buffer and glycerol and allowed tomix completely by stirring. Each ml of the resultant lipid blendcontained 0.75 mg total lipid (consisting of 0.400 mg DPPC, 0.046 mgDPPE, and 0.32 mg MPEG-5000-DPPE). Each ml of the lipid blend alsocontained 103.5 mg propylene glycol, 126.2 mg glycerin, 2.34 mg sodiumphosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasicheptahydrate, and 4.87 mg sodium chloride in Water for Injection. The pHwas 6.2-6.8. The material was provided in sealed vials with a headspacecontaining octafluoropropane (OFP) gas (>80%) with the balance air.

FIG. 6 graphically illustrates the sizing profile of microbubblesproduced by the Definity equivalent standard and MVT-100. Bothformulations have identical lipid concentration and composition with theexception of substitution of DPPE in MVT-100 for DPPA. The microbubblesproduced by the MVT-100 formulation remain stable over time with respectto concentration and size distribution even while suspended in normalsaline. Referring now to FIGS. 7, 8, 9 and 10. Lipid blends containingdifferent mixtures of the lipids DPPC, DPPE, and DPPE-MPEG-5000 weremade with different proportions of the co-solvents propylene glycol(PGOH), glycerol (GLOH), and water containing a sodium phosphate bufferand sodium chloride (H₂O). The recited co-solvent percentage ratios arelisted as volume percent abbreviated as % (v/v). For example 10:10:80,equates to 10% (v/v) propylene glycol, 10% (v/v) glycerol and 80% (v/v)water containing sodium phosphate buffer and sodium chloride. The lipidblends are listed below in table 7.

TABLE 7 Total lipid Vial fill Mol % lipid ratios Vol % cosolvent ratiosconc volumes 82:10:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 0.75mg/mL 1.5 mL 82:10:08 DPPC:DPPE:DPPE-MPEG-5K 15:05:80 PGOH:GLOH:H₂O 0.75mg/mL 1.5 mL 82:10:08 DPPC:DPPE:DPPE-MPEG-5K 20:80 PGOH:H₂O 0.75 mg/mL1.5 mL 77:15:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 0.75 mg/mL1.5 mL 77:15:08 DPPC:DPPE:DPPE-MPEG-5K 15:05:80 PGOH:GLOH:H₂O 0.75 mg/mL1.5 mL 77:15:08 DPPC:DPPE:DPPE-MPEG-5K 20:80 PGOH:H₂O 0.75 mg/mL 1.5 mL72:20:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 0.75 mg/mL 1.5 mL72:20:08 DPPC:DPPE:DPPE-MPEG-5K 15:05:80 PGOH:GLOH:H₂O 0.75 mg/mL 1.5 mL72:20:08 DPPC:DPPE:DPPE-MPEG-5K 20:80 PGOH:H₂O 0.75 mg/mL 1.5 mL82:10:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 0.75 mg/mL 1.0 mL82:10:08 DPPC:DPPE:DPPE-MPEG-5K 15:05:80 PGOH:GLOH:H₂O 0.75 mg/mL 1.0 mL82:10:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 1.00 mg/mL 1.5 mL82:10:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 1.50 mg/mL 1.5 mL77:15:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 1.00 mg/mL 1.0 mL77:15:08 DPPC:DPPE:DPPE-MPEG-5K 10:10:80 PGOH:GLOH:H₂O 1.50 mg/mL 1.0 mL77:15:08 DPPC:DPPE:DPPE-MPEG-5K 15:05:80 PGOH:GLOH:H₂O 1.00 mg/mL 1.0 mL

The lipid blend containing 82 mol % DPPC, 10 mol % DPPE, and 8%DPPE-MPEG-5000 was prepared with both a sodium phosphate buffer as wellas a Histidine-Glutamic Acid buffer. The lipid blend was also preparedwith a sodium phosphate buffer at approximate concentrations of 5 and 25mM. Other buffers approved for use in parenteral formulations with a pKain the 5.8-7.8 range such as citric acid may be used as well. Gasfilling in the final product may be 35% air with 65% perfluoropropane.But in these experiments the vials that we filled using the manifold forintroducing the perfluoropropane were >90% for all samples.

FIG. 7 is a comparison of formulations containing differentconcentrations of co-solvents with the same concentration of lipids (82mol % DPPC, 10 mol % DPPE, 8 mol % DPPE-MPEG-5000). Formulationscontaining PGOH:GLOH:H2O at both 10:10:80 and 15:5:80% (v/v) producedsimilar microbubble concentrations as the Definity Equivalent standardwhich contains PGOH:GLOH:H2O at 10:10:80% (v/v). The formulationcontaining PGOH:H2O at 20:80% (v/v) produced a lower concentration ofmicrobubbles than both the lipid blends containing 10:10:80 and 15:5:80%(v/v) PGOH:GLOH:H2O and the Definity Equivalent standard.

FIG. 8 is a comparison of formulations containing differentconcentrations of co-solvents with the same concentration of lipids (77mol % DPPC, 15 mol % DPPE, 8 mol % DPPE-MPEG-5000). Changing the volumefraction of co-solvents did not significantly affect the concentrationof microbubbles produced by the lipid blend.

FIG. 9 is a comparison of formulations containing differentconcentrations of co-solvents with the same concentration of lipids (72mol % DPPC, 20 mol % DPPE, 8 mol % DPPE-MPEG-5000). Increasing thepercentage of DPPE to 20 mole % decreases the number of microbubblescompared to 10-15 mole % DPPE. Changing the volume fraction ofco-solvents did not significantly affect the concentration ofmicrobubbles produced by the lipid blend.

FIG. 10 is a summary slide of the information shown in FIGS. 7, 8 and 9.

Example 4

Samples of Definity equivalent and MVT-100 lipid blends were prepared asdescribed in Examples #1 and #3. HPLC was used to characterize theconcentrations of DPPC, DPPE-MPEG-5000, DPPA, DPPE and palmitic acid(breakdown product of phospholipids from hydrolysis). Samples werestored at 4° C. and 40° C. and assayed after 31 days. Referring to Table8 and Table 9, the degradation of the 3 lipids contained in the Definityequivalent (DPPA, DPPC, and DPPE-MPEG-5K) were significantly higher thanthe degradation of the 3 lipids contained in the MVT-100 formulation(DPPC, DPPE, DPPE-MPEG-5000). As shown in the bottom of Table 8, none ofthe lipids in the Definity equivalent retain over 90% potency after 31days of storage at 40° C. and only one of the lipids is above 88%potency. By comparison all of the lipids in MVT-100 retain >95% potencyas shown in the bottom of Table 9. This difference in lipid degradationrates is illustrated in FIG. 11.

TABLE 8 Table 4. DEFINITY EQUIVALENT Stability Conc (mg/ml) DPPE- PalmAcid MPEG-5K DPPA DPPC BATCH 1 T = 00 days 0.000 0.282 0.046 0.379 0.0000.283 0.047 0.380 0.000 0.284 0.047 0.384 T = 31 days 0.035 0.242 0.0380.311 0.035 0.251 0.039 0.319 0.035 0.251 0.038 0.319 BATCH 2 T = 00days 0.000 0.283 0.047 0.384 0.000 0.288 0.048 0.387 0.000 0.280 0.0470.384 T = 31 days 0.037 0.238 0.038 0.307 0.037 0.239 0.036 0.297 0.0360.234 0.035 0.312 BATCH 3 T = 00 days 0.000 0.306 0.045 0.420 0.0000.307 0.045 0.429 0.000 0.311 0.044 0.428 T = 31 days 0.031 0.266 0.0310.344 0.031 0.265 0.034 0.341 0.033 0.265 0.032 0.343 % Lipid (T = 31days) BATCH DPPE-MPEG-5K DPPA DPPC 1 87.594 82.525 82.989 2 83.42576.726 79.358 3 86.190 71.973 80.503 Average 85.736 77.075 80.95

TABLE 9 Table 5. MVT-100 Stability Conc (mg/ml) DPPE- Palm Acid MPEG-5KDPPC DPPE BATCH 1 T = 00 days 0.000 0.291 0.404 0.049 0.000 0.291 0.3990.049 0.000 0.290 0.394 0.047 T = 31 days 0.000 0.282 0.377 0.046 0.0000.275 0.385 0.045 0.000 0.283 0.395 0.045 BATCH 2 T = 00 days 0.0000.294 0.409 0.044 0.000 0.288 0.397 0.045 0.000 0.281 0.398 0.044 T = 31days 0.000 0.274 0.386 0.043 0.000 0.273 0.380 0.043 0.000 0.273 0.3860.043 BATCH 3 T = 00 days 0.000 0.278 0.393 0.049 0.000 0.280 0.3940.050 0.000 0.278 0.390 0.049 T = 31 days 0.000 0.269 0.377 0.048 0.0000.268 0.373 0.048 0.000 0.264 0.376 0.049 % Lipid (T = 31 days) BATCHDPPE-MPEG-5K DPPC DPPE 1 96.270 96.653 93.695 2 95.022 95.612 97.347 395.929 95.584 97.554 Average 95.740 95.950 96.199

Example 5

Complement-mediated retention of microbubbles in the renal cortex ishypothesized to be responsible for back/flank pain that occurs as aside-effect of Definity. A study was performed with Difinity equivalentand MVT-100 in wild-type mice. Mice were injected IV with eitherDefinity equivalent (n=10) or MVT-100 (n=10) at a dose of 5×10⁵microbubbles. Ultrasound imaging was performed 8 minutes aftermicrobubbles injection, allowing enough time for blood pool microbubblesto clear. Referring now to FIG. 17, residual microbubbles in the renalcortices were detected on ultrasound. Definity equivalent accumulatedover three-fold more in the renal cortices than MVT-100. The datagraphically shown in FIG. 17 demonstrate that MVT-100 has less renalretention than Definity equivalent, and suggest that MVT-100 should havea lower incidence of back/flank pain than Definity.

As shown in FIG. 12, Definity causes much more delayed renal enhancementthan MVT-100 (Mb-neutr).

Example 6

Echocardiography was performed in 5 pigs. Animals were injected randomlywith either Definity or MVT-100. Ultrasound parameters were frequency=2MHz, MI=0.18 or 0.35. Each vial was mixed in a 100 ml bag of saline.Approximate volume in each vial was 1.5-1.6 ml. All were infused at arate of 3.6 to ˜5.0 mL per minute. Pig weights were ˜27-30 kg.

Assuming 1.5 ml microbubbles/100 ml=15 uLMB/ml solution×3.6 mL/min=54uL/min; divided by 30 kg=1.8 uL/kg/min. Images were assessed by theoperators for contrast enhancement of the heart chambers and myocardium.Animals were monitored for blood pressure, heart rate and paO₂. Imagecontrast was judged to be comparable from MVT-100 and Definity. Therewas no change in heart rate, blood pressure or paO₂ after injection ofeither agent. Imaging was comparable with both MVT-100 and Definity.

Example 7 Cationic Lipid Use

A lipid blend was prepared as in Example 1, comprising a cationic lipid1,2-Distearoyl-3-trimethylammonium-propane chloride 9 (DSTAP) incombination with, inter alia, a cone-shaped neutral lipid MPEG-5K-DPPE.

Table 10 summarizes particle size data for three (3) compositions at twotime points, namely four (4) minutes post-activation and 64 minutespost-activation.

TABLE 10 Post-Act Mean Time Num Vol 0.51-10 μm 10-25 μm >25 μm Sample ID(min) Wt Wt Counts % Counts % Counts % 72:10:10:8 4 0.96 13.79 8.12E+0999.9914 6.67E+05 0.0082 1.30E+05 0.0016 DPPC:DPPE:DSTAP:MPEG-5K-DPPE72:10:10:8 64 1.06 17.09 7.37E+09 99.9440 5.26E+06 0.0713 1.16E+050.0016 DPPC:DPPE:DSTAP:MPEG-5K-DPPE 77:5:10:8 4 0.93 156.32 8.08E+0999.9912 6.81E+05 0.0084 1.30E+05 0.0016 DPPC:DPPE:DSTAP:MPEG-5K-DPPE77:5:10:8 64 1.01 33.80 7.10E+09 99.9725 2.38E+06 0.0335 2.03E+05 0.0029DPPC:DPPE:DSTAP:MPEG-5K-DPPE 82:10:8 4 0.91 15.13 9.75E+09 99.99484.20E+05 0.0043 1.16E+05 0.0012 DPPC:DSTAP:MPEG-5K-DPPE 82:10:8 64 1.0228.18 9.79E+09 99.9201 8.30E+06 0.0847 9.86E+05 0.0101DPPC:DSTAP:MPEG-5K-DPPE SALINE N/A 0.89 32.89 1.94E+07 99.7017 4.35E+040.2237 4.35E+04 0.2237

FIG. 12 graphically illustrates total microbubble counts measured forthe three formulations listed in Table 10+a formulation containing 62mol % DPPC, 10 mol % DPPE, 20 mol % DSTAP, and 8 mol % DPPE-MPEG-5K.Referring to Table 10. The formulation comprising 82 mole percent ofDPPC, 10 mole percent DSTAP, and 8 mole percent MPEG-5K-DPPE showed thegreater number of microbubbles at both four (4) minutes post activationand sixty-four (64) minutes post-activation.

Prophetic Example 8

Thousands of patients are administered Applicant's microbubblecomposition described above in Example 1. Compared to clinical use ofDEFINITY, the incidence of back pain is less using Applicant'smicrobubble composition of Example 2.

Prophetic Example 9

A stability study is performed at room temperature. HPLC is used tomonitor the break-down of the lipids. Samples are periodically agitatedon the VialMix to produce microbubbles. The numbers of microbubbles andsize are studied by a particle sizing system. Applicant's microbubblecomposition of Example 2 has a longer shelf-life at room temperaturethan DEFINITY, also as confirmed in Example 4.

Prophetic Example 10

DPPC, DPPE-PEG(5000) and DPPE in the same ratios as Example 3 aredissolved in chloroform and agitated and heated until dissolved in around bottom flask. The chloroform is evaporated under heat and reducedpressure leaving a dry film of lipids. The lipids are rehydrated in amixture of water containing Macrogol 4000. The material is agitateduntil the lipids are suspended uniformly. The suspension is placed intovials and lyophilized. The vials contain a dried cake of lipid with PEGfilled with head space ofperfluorobutane (PFB) gas and nitrogen (65%PFB/35% nitrogen). The vials are sealed and heated to 38° C. for 4hours. For clinical imaging use, the microbubbles are prepared byinjecting normal saline into the vials and gentle agitation by hand.

Prophetic Example 11

Example 2 is substantially repeated except that one-tenth of theDPPE-PEG(5000) is replaced by DPPE-PEG(5000)-Folate. The resulting lipidsuspension contains 0.75 mg lipid blend (consisting of 0.046 mg DPPE,0.400 mg DPPC, and 0.274 mg MPEG5000 DPPE) and 0.030 mg DPPE (PEG5000)Folate. The lipid suspension is then useful for making microbubbles totarget cells, e.g. cancers, over expressing the folate receptor.Compared to microbubbles containing phosphatidic acid, microbubblesprepared with the above formulation containing DPPE have improvedtargeting and cellular uptake.

Prophetic Example 12

The lipids used in Example 2 are used to emulsify perfluoropentane. Thefinal concentration of perfluoropentane is 2% w/vol and the lipids are 3mg/ml. The chilled material is transferred into vials and the head spaceof air is removed from the vials by negative pressure. The vials aresealed. To produce microbubbles the sealed vials are then agitated on aVialMix as described in Example 2.

Prophetic Example 13

An emulsion of perfluoropentane is prepared usingDPPC/DPPE-PEG(5,000)/DPPE by homogenizing the lipids with DDFP by highpressure homogenization under elevated pressure at 4° C. The resultingemulsion had 2% w/vol DDFP and 0.3% w/vol lipid. A similar emulsion isprepared with DPPC/DPPE-PEG without DPPE. Samples are stored in sealedvials at room temperature. Particle sizing shows increased particlecount and better maintenance of particle size for the formulationcontaining DPPE.

While the preferred embodiments of the present invention have beenillustrated in detail, it should be apparent that modifications andadaptations to those embodiments may occur to one skilled in the artwithout departing from the scope of the present invention as set forthherein.

1-17. (canceled)
 18. A composition for stabilizing a fluorocarbonemulsion, comprising: a fluorocarbon selected from perfluorobutane andperfluoropentane; phosphatidylcholine;phosphatidylethanolamine-polyethylene glycol (PEG); anddipalmitoylphosphatidylethanolamine (DPPE), wherein the compositioncomprises no dipalmitoylphosphatidic acid (DPPA), the composition has apH between 6.2 to 6.8, and the dipalmitoylphosphatidylethanolamine(DPPE) is at a concentration between about 5 and about 20 mole percentof the composition.
 19. A lyophilized composition, comprising: afluorocarbon selected from perfluorobutane and perfluoropentane;phosphatidylcholine; phosphatidylethanolamine-polyethylene glycol (PEG);and dipalmitoylphosphatidylethanolamine (DPPE), wherein the compositioncomprises no dipalmitoylphosphatidic acid (DPPA), the composition has apH between 6.2 and 6.8, and the dipalmitoylphosphatidylethanolamine(DPPE) is at a concentration between about 5 and about 20 mole percentof the composition.
 20. A composition of a fluorocarbon emulsion,comprising: a fluorocarbon selected from perfluorobutane andperfluoropentane; phosphatidylcholine;phosphatidylethanolamine-polyethylene glycol (PEG); anddipalmitoylphosphatidylethanolamine (DPPE), wherein the composition hasa pH between 6.2 to 6.8, and the dipalmitoylphosphatidylethanolamine(DPPE) is at a concentration between about 5 and about 20 mole percentof the composition.
 21. The composition of claim 18, wherein thefluorocarbon comprises perfluorobutane.
 22. The composition of claim 18,wherein the fluorocarbon comprises perfluorobutane.
 23. The compositionof claim 18, wherein the fluorocarbon comprises perfluoropentane andperfluoropentane.
 24. The lyophilized composition of claim 19, whereinthe fluorocarbon comprises perfluorobutane.
 25. The lyophilizedcomposition of claim 19, wherein the fluorocarbon comprisesperfluorobutane.
 26. The lyophilized composition of claim 19, whereinthe fluorocarbon comprises perfluoropentane and perfluoropentane. 27.The composition of claim 20, wherein the fluorocarbon comprisesperfluorobutane.
 28. The composition of claim 20, wherein thefluorocarbon comprises perfluorobutane.
 29. The composition of claim 20,wherein the fluorocarbon comprises perfluoropentane andperfluoropentane.